Association of IL-17A Polymorphism with Chronic Periodontitis in Type 1 Diabetic Patients

Statement of the Problem: The association of genetic polymorphisms with periodontitis has been studied extensively. The interleukin -17 (IL-17) is a group of cytokines, which comprises six different molecules (IL-17A, B, C, D, E & F). Among these, IL-17A & F are the most commonly understood cytokine, which plays a critical role in inflammatory diseases and periodontal inflammation. Purpose: To evaluate whether IL-17A gene polymorphism is associated with increased risk of chronic periodontitis in type 1 diabetes patients. Materials and Method: This quantitative case- control study was carried out in 60 subjects in 4 groups. The study groups included group A: 15 type 1 diabetes patients (T1DM) with chronic periodontitis (CP), group B: 15 T1DM patients without CP, group C: 15 Non-diabetic patients with CP, group D: 15 Non-diabetic patients without CP. Blood samples were drawn from the subjects and analyzed for IL-17A polymorphism by using the polymerase chain reaction-restriction fragment length polymorphism method. Results: There was no statistical significant difference seen in the genotype distribution among CP patients with or without T1DM and healthy controls. Odds ratio and p value indicated that increased risks for CP were associated with IL-17A allele (G) in patients with T1DM. This allele was correlated with worse clinical parameters of CP in T1DM patients. Conclusion: The present study revealed that IL-17A (rs2275913) polymorphism was not associated with increased risk for CP in T1DM patients.


Introduction
Chronic Periodontitis is a complex infectious-inflammatory disease, immune response play a major role in irreversible destructions of gingival tissue, periodontal ligament, and alveolar bone destruction [1]. The periodontal pathogens initiate the activation of innate and adaptive immunities, which leads to the release of inflammatory mediators from host tissues [2]. This excessive production of inflammatory mediators triggers the tissue destruction and provokes further progression of the disease. This progression can be attributed to the more production of pro-inflammatory cytokines and decreased amount of anti-inflammatory mediators [3].
The main factors in the immunoregulation of periodontal disease depend on the controlled balance between the T helper 1 & 2 (Th 1/Th2) cells [4].
Among the T-helper cells, Th17 subset is recently established prominent focus, because of its role in the autoimmune disease pathogenesis. The interleukin -17  consists of six groups of cytokines (IL-17A to IL-17F) and is expressed by the CD4 + T-helper subset (T-helper 17 lineage) and mediates the tissue inflamma-tion [5]. This causes the activation of T cells, fibroblasts, osteoclast, and the maturation of dendritic cells. Reports indicate that IL-17A has a key role in the progression of chronic and aggressive periodontitis [6][7]. IL-17A triggers the proinflammatory cytokines such as TNF-α, IL-6, and IL-1β expressions and promotes neutrophil migration. It can facilitate the function of CD 8 + T cells and causes the B cell activation to produce antibodies. These indicated that IL-17A cytokines play a role in potentially promoting inflammatory and periodontal disease [8].
IL-17 cytokines are also involved in the initiation of autoimmunity in rheumatoid arthritis, experimental autoimmune encephalomyelitis, multiple sclerosis, systemic lupus erythematosus, and auto-immune diabetes (T1DM) [9][10][11][12]. The risks of T1DM are ALA-associated genotypes and B cell autoantibodies. Recently, the role of T helper 17 (Th 17) cells, which secrete IL-17 in pathogenicity, was also suggested as a risk factor in T1DM development [13]. IL-17 gene is located on each side of the human chromosome 6p12.2. IL-17 shares 50% amino acid with chromosome 6 and similar functions (almost 50%) as that of chromosome 6. In vitro study revealed that in diabetic patients, there was increased expression of IL-17A, which was primarily induced by monocytes [14]. IL-17 producing cells were commonly found in peripheral blood and among the monocytes of chronic T1DM .The activation of this IL-17 pathway accelerates the apoptosis of pancreatic β cells, leading to T1DM [15]. The studies done on two different groups of children with new onset of T1DM revealed that there was increase in number of IL-17 producing T-cells in their peripheral blood [16].
IL-17A cytokine in periodontal diseases was significant associated with disease progression and severity of destruction. IL-17A levels were also significantly elevated in saliva and gingival crevicular fluids of patients with chronic periodontitis [17][18]. IL-17A polymorphism was studied in the literature; it was found to be commonly associated with diabetes, and chronic and aggressive type of periodontal disease [19][20]. The single nucleotide polymorphisms (SNP) in the lL-17 gene had important effect on the production of IL-17 in T1DM. In addition, Linhartova et al. [19] showed that the correlation between increased levels of IL-17 and A allele of 197A/G SNP16.
The role of this A allele was also marginally associated with the increased risk of T1DM and CP. The mechanism of IL-17 in human T1DM provides a new view on the pathogenesis of the disease with periodontitis and implies a novel potential therapeutic strategy in T1DM by controlling the role of IL-17. There were many studies showing a contradictory role of Th17 in both protection and pathogenesis of T1DM and CP [17][18][19][20]. Hence, this study was to done to evaluate the association of IL-I 7A polymorphism in CP with T1DM patients.

Materials and Method
The study was taken out in the outpatient Department of which includes presence of clinical attachment loss 3-4mm, pocket probing depth (PPD) of 5mm at least more than 30% teeth involved, and 15% to 30% coronal third radiographic bone loss [21]. Exclusion criteria were smokers, ongoing orthodontic therapy, aggressive periodontitis, general health problems (hepatitis, HIV infection, and chemotherapy), pregnancy, lactation, and non-Indian races. Periodontal parameters were measured using Williams probe and the gingival Index was measured for assessing the severity of gingivitis [22]. Diabetic parameters were monitored with HbAlc and fasting blood glucose levels (FBS). HbA1c value of <7% revealed there was sufficiently good control of blood glucose in T1DM patients and FBS was monitored with cutoff points of > 120mg/dl [23].

DNA Extraction
The venous blood (5ml) was collected from cubital fossa of the patients under strict sterile conditions and transferred to the laboratory in a falcon tube containing ethylenediaminetetraacetic acid (EDTA) [16]. It was stored at -200 o C for DNA separation. The 200μL of the blood sample was placed into a micro centrifuge tube and 600μL of RBC cell lysis solution was added to the blood sample. The uniformity in the sample mix was obtained by inverting the tube several times and flicking the bottom of the tube. The incubation of the sample was done for 5 minutes at room temperature and shaken briefly.
Again, it was repeated at room temperature for 5 minutes,

DNA Amplification by Polymerase Chain Reaction
The primer pair (Bioserve, Beltsville, USA) used for this study to check IL-l7A polymorphism was, Sense: The amplification was done using conventional PCR

Results
In total, 60 cases (35 females, 25 males) aged between 30-44 years were included in the present study. The comparisons between groups as well as periodontal status are given in Table 1 (Table 4). When considering the dominant and recessive models, the patients with CP (group C) had a statistical significant difference when compared (A+G versus G) with a healthy group (p= 0.120) ( Table 2).

Discussion
Previous studies in the literature reported that IL-17A polymorphism with AA (dominant) genotype and A allele were commonly associated with type 1 and 2 diabetes and CP than GG (recessive) genotype and G allele [24][25]. In order to find the genotype and allele association, we evaluated IL-17A rs2275913 polymorphism in a group of patients with and without T1DM and/or CP from South Indian population.
In this study, AA, AG, and GG genotype were asse-   There are some limitations in this study, which are described as follows. First, the study was done with small sample size of patients included in each group.
Secondly, uncontrolled diabetes patients were not included for in this study. This could lead to the genotypic and allelic variation bias in the IL-17A polymorphism.
Third, the patients were selected from the same zone of location; this could mislead the genetic variation.
Fourth, the diabetes and non-diabetes groups were not age-matched. However, within the limitations, the study showed no association of IL-I 7A polymorphism in T1DM with CP patients that indicates the possible importance in disease pathology of chronic periodontitis.

Conclusion
The present study demonstrated no association of IL-17A polymorphism in CP with T1DM patients of the south Indian population. Further studies with large sample size might give an evidence-based overview of IL-17A polymorphisms and CP. In addition, there was a genotype similarity among the patients and controls; we hypothesized that IL-I 7A is not a polymorphic within our Indian population.

Conflicts of Interest
None declared.